Expression of Enzymatically Active CYP3A4 by Caco-2 Cells Grown on Extracellular Matrix-Coated Permeable Supports in the Presence of 1α,25-Dihydroxyvitamin D₃

Abstract

The human colon carcinoma cell line, Caco-2, is widely used as a model for oral absorption of xenobiotics. The usefulness of Caco-2 cells has been limited, however, because they do not express appreciable quantities of CYP3A4, the principle cytochrome P450 present in human small bowel epithelial cells. We report that treatment of Caco-2 cells with 1 alpha,25-dihydroxyvitamin D3, beginning at confluence, results in a dose- and duration-dependent increase in CYP3A4 mRNA and protein, with little apparent effect on the expression of CYP3A5 or CYP3A7. This treatment also results in increases in NADPH cytochrome P450 reductase and P-glycoprotein (the MDR1 gene product) but has no detectable effect on expression of CYP1A1, CYP2D6, cytochrome b5, liver or intestinal fatty acid binding proteins, or villin. Maximal expression of CYP3A4 requires an extracellular matrix on a permeable support and the presence of serum. In the treated cells, the intrinsic formation clearance of 1'-hydroxymidazolam (a reaction characteristically catalyzed by CYP3A enzymes) was estimated to be somewhat lower than that of human jejunal mucosa (1.14 and 3.67 ml/min/g of cells, respectively). The 1'-OH-midazolam/4-OH-midazolam product ratio produced by the cells (approximately 5.3) is comparable to, but somewhat lower than, that observed in human jejunal microsomes (7.4-15.4), which may reflect the presence of CYP3A7 in the Caco-2 cells. 25-Hydroxyvitamin D3 is less efficacious but reproduces the effects of the dihydroxy compound, whereas unhydroxylated vitamin D is without appreciable effect. These observations, together with the time course of response, suggest that the vitamin D receptor may be involved in CYP3A4 regulation. The culture model we describe should prove useful in defining the role of CYP3A4 in limiting the oral bioavailability of many xenobiotics.

Expression of CYP3A4, CYP2B6, andCYP2C9 Is Regulated by the Vitamin D Receptor Pathway in Primary Human Hepatocytes

“…In an exploratory part of this work, we found that 1␣,25-(OH) 2 D 3 is an inducer of CYP3A4 in human hepatocytes, as previously observed by others in intestinal cell lines (16,17).…”

“…Both NR1 and NR2 are imperfect DR4 motifs and essential for phenobarbital induction of CYP2B genes. In human CYP2B6, the phenobarbital-responsive element module is located between -1684 and -1733 and has been shown to bind to and be transactivated by CAR and by PXR (12, 15).Previous reports revealed that 1␣,25-dihydroxyvitamin D 3 (1␣,25-(OH) 2 D 3 ), the most active metabolite of vitamin D 3 , behaves as a transcriptional inducer of CYP3A4 in the colic carcinoma Caco-2 cell line and in the human intestinal LS180 cell line (16,17). Vitamin D 3 function is mediated through the vitamin D receptor (VDR; NR1I1), which, after binding 1␣,25-(OH) 2 D 3 with high affinity, forms heterodimers with .…”

“…Previous reports revealed that 1␣,25-dihydroxyvitamin D 3 (1␣,25-(OH) 2 D 3 ), the most active metabolite of vitamin D 3 , behaves as a transcriptional inducer of CYP3A4 in the colic carcinoma Caco-2 cell line and in the human intestinal LS180 cell line (16,17). Vitamin D 3 function is mediated through the vitamin D receptor (VDR; NR1I1), which, after binding 1␣,25-(OH) 2 D 3 with high affinity, forms heterodimers with RXR (18 -20).…”

Involvement of the CYP1A Subfamily in Stereoselective Metabolism of Carvedilol in .BETA.-Naphthoflavone-Treated Caco-2 Cells

“…In addition, we evaluated the expression of mRNA of CYP1A1 in Caco-2 cells, since CYP1A1 is expressed in the cells. 18) Figure 1 shows the CYP/GAPDH ratio of mRNA in non-treated, VD 3 -treated, and b-NF-treated Caco-2 cells. In non-treated Caco-2 cells, the expression of CYP1A1 mRNA was greater than that of any other CYP mRNA.…”

Identification of human UDP-glucuronosyltransferases catalyzing hepatic 1α,25-dihydroxyvitamin D3 conjugation

“…Much of this variability results from differences in CYP3A4 protein content, which can vary more than 8-fold even in mucosal epithelium obtained from healthy volunteers [1,2]. Although the source of that variability is probably multi-factorial, some studies have documented dynamic changes in CYP3A4 transcription that can occur following activation of the vitamin D receptor (VDR) by its natural ligand, 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) [3,4,5,6]. The intestinal mucosa is an important target tissue for 1,25(OH) 2 D 3 , where it regulates the expression of calcium binding and transport proteins and helps maintain calcium homeostasis throughout the body [7,8,9].…”